Despite widespread agreement that the basic unit of function of the T cell arm of the cellular immune system is the collection of memory/effector T cell clones responsive to a specific antigen (Ag), there is very little understanding of the development and homeostasis of Ag-specific human memory/effector T cells in either health or disease. In large part, this lack of definitive information is methodological; i.e. the simple lack of tools to reliably quantitate and functionally evaluate Ag-specific T cells in the human. For many years, assays of antigen specific T cell function relied on proliferation of cultured PBMC as the measure of the responding T cells.
New knowledge of immunological processes in the past decade, however, have highlighted serious problems with this approach. Proliferative responses require days to manifest, and it is now well documented that many, if not most, memory/effector T cells respond to Ag with an effector response (e.g. cytokine release or cytotoxicity) and then succumb to activation-induced apoptosis prior to any possible proliferative response. Those cells that survive to proliferate are thus a highly variable subset (depending on culture conditions) of the overall Ag-responsive cohort. This issue is even more critical for the evaluation of T function in diseases such as AIDS, as it has been clearly documented that memory/effector T cells from HIV+ individuals display an increased susceptibility to apoptosis, and indeed, show enhanced Ag-induced apoptosis in vitro . Similarly T cell responses to antigen in other diseases may be characterized by varying ratios of cells undergoing proliferation, cytokine expression, apoptosis, or cytotoxic T cell differentiation.
Recently, other assays of T cell function have also included cytokine release endpoints, a defining function of memory/effector T cells. While this addition to the measurement of proliferative responses represents a clear advance, in terms of a more comprehensive measure of T cell activation, these assays do not define the functional cell types that participate in these responses. Further, most of these analyses have relied on ELISA-based quantitation of expressed levels of cytokine in culture supernatants. These procedures yield only bulk measurements; individual cell types participating in the response are not evaluated.
To assess which cell types are expressing selected cytokines, subset purification or single cell-based methodologies such as the ELISPOT and cytokine-release limiting dilution assays have been employed. However, these assays are extremely laborious—especially if set up to measure the multiple, potentially relevant cytokines which may be produced by functionally heterogeneous Ag-specific memory/effector T cells—and therefore are not amenable to routine use in the clinical laboratory. Moreover, the sensitivity of these assays may be limited by (1) conditions which do not mimic in vivo cellular environments which may be more clinically relevant, (2) T cell stimulation conditions that must necessarily compromise between conditions optimal for T cell activation and those compatible with the single cell detection strategy of the assay (e.g. limiting dilution conditions or spot counting on nitrocellulose covered culture wells), and (3) vulnerability to some loss of Ag-reactive cells from activation-induced apoptosis (as these assays require from 1-2 to many days of in vitro culture, and activation-induced apoptosis, as measured by DNA strand breaks, can initiate in as few as 12-19 hours).
It has been previously shown that T cell cytokine responses occur in cells expressing the CD69 antigen. A TH1, or type I response, for example is typically identified as the percentage of CD3+ T cells co-expressing CD69 and γ-IFN, IL-2, or TNF-α. A TH2, type II, response can be characterized as the frequency of CD3+ T cells which co-express CD69, and IL-4 or IL-13. The distribution of individual cytokines of a type I or type II response may also be descriptive of a specific kind of cellular response to antigen. For example unique T cell subsets may express TNF-α and γ-IFN in response to different antigens and have been referred to as TH1 cells.